Harnessing phage-ciprofloxacin synergy to overcome multidrug-resistant infections and biofilm persistence.

Enterococcus faecalis is a Gram-positive opportunistic pathogen commonly associated with hospital-acquired and chronic wound infections. Its capacity for biofilm formation and increasing resistance to standard antibiotics, particularly ciprofloxacin (CIP), complicates treatment. The emergence of CIP-resistant E. faecalis (EFR) strains limits existing antibiotic options and requires alternative treatment approaches. Our study evaluated the antibacterial efficacy of a lytic bacteriophage (φR12EFP) and its combination with CIP against E. faecalis. Phage φR12EFP was isolated from environmental samples and characterized. Transmission electron microscopy revealed an icosahedral tailed morphology. Whole-genome sequencing identified a 58.6 kb linear double-stranded DNA genome with high coding density and no lysogeny-associated genes, confirming its classification within the Saphexavirus genus. φR12EFP exhibited host specificity toward E. faecalis. Growth curve analysis showed the emergence of bacterial resistance after 10 h of phage exposure. Adsorption and one-step growth assays demonstrated rapid host attachment, with a burst size of approximately 356 PFU/cell, and optimal antibacterial activity at a multiplicity of infection (MOI) of 0.1. φR12EFP remained stable between 4 and 60 °C and pH 5-9. A CIP-resistant EFR strain was developed under selective pressure to establish a clinically relevant model. Combined treatment with φR12EFP (MOI 0.1) and low-dose CIP (5 µg/mL) exhibited EFR bacterial suppression and restored antibiotic efficacy. The combination reduced established EFR biofilm by 75% and improved embryo survival to 90-100% in an in ovo infection model compared with φR12EFP monotherapy alone. These findings indicate that φR12EFP restores CIP sensitivity in resistant E. faecalis and supports the use of phage-antibiotic regimens against multidrug-resistant enterococci.